The purpose of this study was to determine simply and accurately ploidy levels as estimated by changes in nuclear DNA content of wheat (Triticum aestivum L.) plants regenerated from microspore-derived embryos. Using flow cytometry, the nuclear DNA content of green (83) and albino (222) plants derived using anther culture of ‘Bobwhite’ and ‘Pavon 76’, and of their reciprocal F₁ hybrids was estimated. The average DNA content of the Bobwhite and Pavon 76 standards was 32.46 and 31.28 per nucleus, respectively. Microspore-derived haploid (3×), doubled-haploid (6×), nanoploid (9×), and dodecaploid (12×) plants contained on average 15.44, 30.56, 45.57, and 60.27 pg of DNA, respectively, at a ratio of 1:1.98:2.99:3.90. The frequency of haploids (43.6%) was similar to that of doubled haploids (43.0%), and much larger than the frequency of endopolyploids [nanoploid (1.3%) and dodecaploid (1.0%)] and various aneuploids (11.1%). In terms of genetic stability, green plants had less chromosomal variation than albino plants. The procedure is suitable for rapid determination of the ploidy levels of wheat microspore-derived plants. The knowledge about DNA content or genome size of plants obtained here provides useful information to plant breeders and geneticists interested in using anther culture.